Current antivirals for influenza infection target specific viral proteins. Due to marked genetic diversity, different strains of influenza demonstrate differential sensitivity to marketed anti-influenza drugs. Additionally, current drugs remain vulnerable to the rapid development of virus resistance. The present project proposes to validate a paradigm-shifting antiviral mechanism-of-action, the activation of host-encoded sirtuins. Sirtuins are a family of seven NAD+-dependent deacylases known for regulating numerous cellular and organismal functions, including metabolism, cell cycle and longevity. Sirtuins may also be evolutionarily conserved broad- spectrum viral restriction factors based on experiments demonstrating that activation of sirtuins in eukaryotic or prokaryotic host cells increases growth of diverse viruses including bacteriophages (Koyuncu et al. 2014, mBio 5:e02249). In the case of influenza A, Sirt1 and Sirt6 have the largest effects on virus growth. Indeed a small molecule screen for sirtuin agonists identified a Sirt1 and a Sirt1&6 activator, each with a distinct chemical scaffold, as potent broad-spectrum antivirals; completed medicinal chemistry improved the antiviral potency of the Sirt1&6 activator compared to the screen identified molecule and a patent application was filed on this scaffold. In addition, two independently published mouse studies demonstrate in vivo anti-influenza efficacy for two plant polyphenols resveratrol and isoquercetin that are now known to be Sirt1 activators. Importantly, isoquercetin prevented the accumulation of viral resistance observed for direct-acting antivirals amantadine and oseltamivir during serial passage in culture. Proposed Phase I goals are to confirm sirtuin activation provides efficacy against multiple seasonal, pandemic, and resistant influenza A and B strains, and a high barrier against future acquired resistance in cell culture; and to reproduce the apparent antiviral efficacy observed in mouse influenza challenge for proposed proprietary activators as was demonstrated for tool compounds, Sirt1 activators isoquercetin and resveratrol. Once a lead series is prioritized based on the Phase I results, the Phase II component of the grant will further progress a medicinal chemistry campaign to improve the potency and pharmacokinetic properties of the lead series to deliver advanced compounds with oral bioavailability and comparable efficacy as stand-alone or in combination with oseltamivir in the mouse model. Such a product will address unmet medical need compared to oseltamivir, because compared to oseltamivir, these drugs should 1) broadly inhibit all subtypes of influenza A and B; 2) block the replication of viruses resistant to current therapies; and 3) dramatically reduce the development of viral resistance during stand- alone or combination treatment.